Эволюция подходов к манипуляции генетическим материалом живых объектов: от селекции к редактированию генома

Системы редактирования генома представляют собой мощный инструмент, способный прицельно модифицировать генетический материал в его природном контексте внутри живого организма. Благодаря таким удивительным возможностям системы редактирования нашли широкий спектр применения во всех сферах биологической науки и медицины. В данной рукописи мы проводим краткий исторический экскурс по вопросу возникновения и развития различных систем геномного редактирования. Эволюция данного направления сопровождалась грандиозными открытиями и вручением целого ряда Нобелевских премий. Отслеживая логику научной мысли в стремлении понять и модифицировать материальную основу наследственности, данная рукопись ставит своей целью сформировать у начинающих исследователей целостную картину об огромной вселенной (без преувеличения) систем геномного редактирования. Понимание же полноты всего спектра потенциальной активности систем редактирования обязывает исследователей к вдумчивому и ответственному их применению.

1. Dahm R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Hum Genet. 2008;122(6):565–581. DOI: 10.1007/s00439-007-0433-0

2. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953;171(4356):737–738. DOI: 10.1038/171737a0

3. Lehnert H, Berner T, Lang D, Beier S, Stein N, Himmelbach A, Kilian B, Keilwagen J. Insights into breeding history, hotspot regions of selection, and untapped allelic diversity for bread wheat breeding. Plant J. 2022;112(4):897–918. DOI: 10.1111/tpj.15952

4. Muller HJ. Artificial transmutation of the gene. Science. 1927;66(1699):84–87. DOI: 10.1126/science.66.1699.84

5. Riaz M, Yasmeen E, Saleem B, Hameed MK, Saeed Almheiri MT, Saeed Al Mir RO, et al. Evolution of agricultural biotechnology is the paradigm shift in crop resilience and development: a review. Front Plant Sci. 2025;16:1585826. DOI: 10.3389/fpls.2025.1585826

6. Miescher F. Ueber die chemische Zusammensetzung der Eiterzellen. Hoppe-Seyler’s Med Chem. 1871;4:441–460.

7. Altmann R. Die Elementarorganismen und ihre Beziehungen zu den Zellen. Leipzig: Veit & Co., 1890.

8. Griffith F. The significance of pneumococcal types. J Hyg (Lond). 1928;27(2):113–159. DOI: 10.1017/S0022172400031879

9. Avery OT, MacLeod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation. J Exp Med. 1944;79(2):137–158. DOI: 10.1084/jem.79.2.137

10. Todd AR. Nucleotides, nucleosides, and nucleic acids. Angew Chem. 1958;70(1):1–17. DOI: 10.1002/anie.195800011

11. Chargaff E. Chemical specificity of nucleic acids and mechanism of their enzymatic degradation. Experientia. 1950;6(6):201–209. DOI: 10.1007/BF02173653

12. Franklin RE, Gosling RG. Molecular configuration in sodium thymonucleate. Nature. 1953; 171:740–741. DOI: 10.1038/171740a0

13. Hershey AD, Chase M. Independent functions of viral protein and nucleic acid in the growth of bacteriophage. J Gen Physiol. 1952;36(1):39–56. DOI: 10.1085/jgp.36.1.39

14. Fraenkel-Conrat H, Singer B. Reconstitution of infectivity with ribonucleic acid and virus protein. Proc Natl Acad Sci USA. 1957;43(9):707–713. DOI: 10.1073/pnas.43.9.707

15. Watson JD, Crick FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737–738. DOI: 10.1038/171737a0

16. Gamow G. Possible relation between deoxyribonucleic acid and protein structures. Nature. 1954;173:318. DOI: 10.1038/173318a0

17. Nirenberg MW, Matthaei JH. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA. 1961;47(10):1588–1602. DOI: 10.1073/pnas.47.10.1588

18. Karagyaur MN, Rubtsov YP, Vasiliev PA, Tkachuk VA. Practical recommendations for improving efficiency and accuracy of the CRISPR/Cas9 genome-editing system. Biochemistry (Moscow). 2018;83(6):629–642. DOI: 10.1134/S0006297918060020

19. Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975;94(3):441–448. DOI: 10.1016/0022-2836(75)90213-2

20. Roberts RJ. Restriction enzymes and their use in molecular cloning. Annu Rev Biochem. 1976;45:485–528. DOI: 10.1146/annurev.bi.45.070176.002501

21. Jackson DA, Symons RH and Berg P. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli Proceedings of the National Academy of Sciences, Vol. 69, No. 10, 1972, pp. 2904-2909. DOI:10.1073/pnas.69.10.2904

22. Mullis K, Faloona F. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. DOI: 10.1016/0076-6879(87)55023-6

23. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, et al. T lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science. 1995;270(5235):475–480. DOI: 10.1126/science.270.5235.475

24. Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab. 2003;80(1–2):148–158. DOI: 10.1016/j.ymgme.2003.08.016

25. Capecchi MR. Altering the genome by homologous recombination. Science. 1989;244(4910): 1288–1292. DOI: 10.1126/science.2660260

26. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51(3):503–512. DOI: 10.1016/0092-8674(87)90646-5

27. Smithies O. The integration of homologous DNA sequences in mammalian chromosomes. Nature. 1985;317(6039):230–234. DOI: 10.1038/317230a0

28. Trudeau DL, Smith MA, Arnold FH. Innovation by homologous recombination. Curr Opin Chem Biol. 2013;17(6):902–909. DOI: 10.1016/j.cbpa.2013.10.007

29. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987;330(6148):576–578. DOI: 10.1038/330576a0

30. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300(5620):763. DOI: 10.1126/science.1078395

31. Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994;14(12):8096–8106. DOI: 10.1128/MCB.14.12.8096-8106.1994

32. Choulika A, Perrin A, Dujon B, Nicolas JF. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol. 1995;15(4):1968–1973. DOI: 10.1128/MCB.15.4.1968-1973.1995

33. Carroll D. Genome engineering with zinc-finger nucleases. Genetics. 2011;188(4):773–782. DOI: 10.1534/genetics.111.131433

34. Roberts RJ. Restriction endonucleases. CRC Crit Rev Biochem. 1976;4:123–164. DOI: 10.3109/10409237609105456

35. Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges. Curr Gene Ther. 2011;11(1):11–27. DOI: 10.2174/156652311794520111

36. Stoddard BL. Homing endonuclease structure and function. Q Rev Biophys. 2005;38(1):49–95. DOI: 10.1017/S0033583505004063

37. Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 2006;34(22):e149. DOI: 10.1093/nar/gkl720

38. Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc Natl Acad Sci USA. 1996;93(3):1156–1160. DOI: 10.1073/pnas.93.3.1156

39. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435(7042):646–651. DOI: 10.1038/nature03556

40. Carroll D. Genome engineering: Zinc-finger nucleases and beyond. Nat Protoc. 2011;6(2): 239–254. DOI: 10.1534/genetics.111.131433

41. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014;15(5):321–334. DOI: 10.1038/nrg3686

42. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009;326(5959):1509–1512. DOI: 10.1126/science.1178811

43. Карагяур МН, Примак АЛ, Джауари СС, Бозов КД, Макусь ЮВ. Технологии редактирования генома и перспективы их применения в биомедицине. Регенерация органов и тканей. 2024;2(1):54–77. DOI: 10.60043/2949-5938-2024-1-54-77

44. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49–55. DOI: 10.1038/nrm3486

45. Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae. 2014;6(3):19–40.

46. Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096. DOI: 10.1126/science.1258096

47. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709–1712. DOI: 10.1126/science.1138140

48. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169(12):5429–5433. DOI: 10.1128/JB.169.12.5429-5433.1987

49. Mojica FJM, Ferrer C, Juez G, Rodríguez-Valera F. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol. 1995;17(1):85–93. DOI: 10.1111/j.1365-2958.1995.mmi_17010085.x

50. Jansen R, van Embden JDA, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565–1575. DOI: 10.1046/j.1365-2958.2002.02839.x

51. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821. DOI: 10.1126/science.1225829

52. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–823. DOI: 10.1126/science.1231143

53. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med. 2021;384(3):252–260. DOI: 10.1056/NEJMoa2031054

54. Nobel Foundation. The Nobel Prize in Chemistry 2020: Emmanuelle Charpentier and Jennifer A Doudna. NobelPrize.org. 2020.

55. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262–1278. DOI: 10.1016/j.cell.2014.05.010

56. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49–55. DOI: 10.1038/nrm3486

57. Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018;9:1911. DOI: 10.1038/s41467-018-04252-2

58. Dyikanov DT, Vasiluev PA, Rysenkova KD, Aleksandrushkina NA, Tyurin-Kuzmin PA, Kulebyakin KY, et al. Optimization of CRISPR/Cas9 technology to knock out genes of interest in aneuploid cell lines. Tissue Eng Part C Methods. 2019;25(3):168–175. DOI: 10.1089/ten.TEC.2018.0365

59. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149–157. DOI: 10.1038/s41586-019-1711-4

60. Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, et al. Structure and Engineering of Francisella novicida Cas9. Cell. 2016;164(5):950–961. DOI: 10.1016/j.cell.2016.01.039

61. Faure G, Saito M, Wilkinson ME, Quinones-Olvera N, Xu P, Flam-Shepherd D, et al. TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses. Science. 2025;388(6746):eadv9789. DOI: 10.1126/science.adv9789

62. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529(7587):490–495. DOI: 10.1038/nature16526

63. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21(2):121–131. DOI: 10.1038/nm.3793

64. Gao C. Genome editing in crops: from bench to field. Nat Rev Mol Cell Biol. 2021;22(7):476–495. DOI: 10.1038/s41580-021-00409-y

65. Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol. 2016;34(1):78–83. DOI: 10.1038/nbt.3439

66. Dipaola MG, Fortuna C, Severini F, Bevivino G, Di Luca M, Nolan T, et al. Temporal and spatial profiling of Aedes albopictus immune responses to chikungunya virus infection. PLoS Negl Trop Dis. 2025;19(10):e0013588. DOI: 10.1371/journal.pntd.0013588

67. Esvelt KM, Smidler AL, Catteruccia F, Church GM. Concerning RNA-guided gene drives for the alteration of wild populations. eLife. 2014;3:e03401. DOI: 10.7554/eLife.03401

68. Mazloum A, Karagyaur M, Chernyshev R, van Schalkwyk A, Jun M, Qiang F, Sprygin A. Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies. Front Vet Sci. 2023;10:1180621. DOi: 10.3389/fvets.2023.1180621

69. Petraitytė G, Preikšaitienė E, Mikštienė V. Genome Editing in Medicine: Tools and Challenges. Acta Med Litu. 2021;28(2):205–219. DOI: 10.15388/Amed.2021.28.2.8

70. Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR–Cas9. Science. 2017;357(6357):1303–1307. DOI: 10.1126/science.aan4187

71. Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 2020;367(6481):eaba7365. DOI: 10.1126/science.aba7365

72. Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, et al. CRISPR–Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med. 2021;385(6):493–502. DOI: 10.1056/NEJ-Moa2107454

73. Intellia Therapeutics. NTLA-2001 phase III clinical data release. 2024.

74. Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J. Don’t edit the human germ line. Nature. 2015;519(7544):410–411. DOI: 10.1038/519410a

75. Baylis F. Altered inheritance: CRISPR and the ethics of human genome editing. CRISPR J. 2019;2(4):203–209. DOI: 10.4159/9780674241954

76. Cornu TI, Mussolino C, Cathomen T. Refining strategies to translate genome editing to the clinic. Nat Med. 2017;23(4):415–423. DOI: 10.1038/nm.4313

77. Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, et al. A prudent path forward for genomic engineering and germline gene modification. Science. 2015;348(6230):36–38. DOI: 10.1126/science.aab1028

78. Raje N, Berdeja J, Lin Y, Siegel D, Jagannath S, Madduri D, et al. Anti–BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N Engl J Med. 2019;380(18):1726–1737. DOI: 10.1056/NEJMoa1817226

79. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR– Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765–771. DOI: 10.1038/nbt.4192

80. Papathanasiou S, Markoulaki S, Blaine LJ, Leibowitz ML, Zhang CZ, Jaenisch R, et al. Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing. Nat Commun. 2021;12(1):5855. DOI: 10.1038/s41467-021-26097-y

81. Uddin F, Rudin CM, Sen T. CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. Front Oncol. 2020;10:1387. DOI: 10.3389/fonc.2020.01387

82. Rehman SU, Abbas GH. CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions. Medicine (Baltimore). 2026;105(2):e47114. DOI: 10.1097/MD.0000000000047114

83. Doudna JA. The promise and challenge of therapeutic genome editing. Nature. 2020; 578(7794):229–236. DOI: 10.1038/s41586-020-1978-5

84. National Academies of Sciences, Engineering, and Medicine. Human Genome Editing: Science, Ethics, and Governance. Washington, DC: National Academies Press; 2017. DOI: 10.17226/24623

85. Karagyaur MN, Efimenko AYu, Makarevich PI, Vasiluev PA, Akopyan ZhA, Bryzgalina EV, et al. Ethical and legal aspects of using genome-editing technologies in medicine (review). Contemporary Technologies in Medicine. 2019;11(3):117–135. DOI: 10.17691/stm2019.11.3.16

86. Bredenoord AL, Schneider M, van Delden JJM. Ethical issues in genome editing: CRISPR– Cas9 and beyond. EMBO Rep. 2021;22(1):e52018. DOI: 10.15252/embr.202052018

87. Bosley KS, Botchan M, Bredenoord AL, Carroll D, Charo RA, Charpentier E, et al. CRISPR germline engineering: the community speaks. Nat Biotechnol. 2015;33(5):478–486. DOI: 10.1038/nbt.3227

88. Wang H, Lu H, Lei YS, Gong CY, Chen Z, Luan YQ, et al., Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects. Mol Ther Methods Clin Dev. 2020;18:390-401. DOI: 10.1016/j.omtm.2020.06.012

89. Xu CL, Ruan MZC, Mahajan VB, Tsang SH. Viral Delivery Systems for CRISPR. Viruses. 2019;4;11(1):28. DOI: 10.3390/v11010028.

90. Park SH, Lee CM, Dever DP, Davis TH, Camarena J, Srifa W, et al. Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease. Nucleic Acids Res. 2019;47(15):7955–7972. DOI: 10.1093/nar/gkz475

91. Hoban MD, Cost GJ, Mendel MC, Romero Z, Kaufman ML, Joglekar AV, et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood. 2016;127(7):839–848. DOI: 10.1182/blood-2015-11-679381

92. Bischof J, Hierl M, Koller U. Emerging Gene Therapeutics for Epidermolysis Bullosa under Development. Int J Mol Sci. 2024;25(4):2243. DOI: 10.3390/ijms25042243